| Objective:In this study,the infected population of dengue fever in Jinghong City,Xishuangbanna in 2019 was analyzed,and the full-length gene sequences extracted and sequenced from the sera of dengue fever patients infected with DENV-1 type were performed for evolutionary analysis,to trace the source of dengue virus epidemic strains.At the same time,wild DENV-1 and DENV-2 strains were isolated from patients' serum and attenuated by gradient cooling culture,and the amino acid mutation sites causing attenuated were obtained by sequencing.The changes of virulence of attenuated and wild strains adapted to low temperature culture were verified by AG 129 infant mouse model.Methods:The number of dengue fever infections in Jinghong City,Xishuangbanna,Yunnan Province in 2019 was statistically analyzed.Then,the RAXML algorithm was used to draw the evolutionary tree of representative dengue virus strains from 1982 to 2020 in China.In addition,the full-length sequence of dengue virus extracted from the sera of dengue patients in Jinghong City,Banna Prefecture was analyzed.C6/36 cells were used to isolate dengue wild virus strains from clinical patient serum,which are susceptible to the virus and extracted from mosquitoes,and transferred the wild virus to Vero cells for purification after adaptive culture.The purified virus strain was gradually cooled from 37? to 28? for attenuated culture,and the virus strains with stable lesions on the cells were retained.The cooling candidate attenuated strain,DENV-1 and DENV-2,were obtained.Full-length gene sequencing of candidate attenuated strains were executed and identified amino acid mutation sites by gene sequence alignment of wild strains.Virulence and concentration of Vero virus were determined by TCID50 of BHK21 cells assisted by Vero virus plaque.The concentrated and purified virus was diluted and injected subcutaneously into two-week-old AG 129(IFN-???R-/-)young mice,and the changes in daily body temperature and body weight were measured.One young mouse was sacrificed every two days for relative quantitative determination of viral load by Q-PCR and pathological analysis of its spleen,liver and brain.The liver,spleen and brain of the young mice were sacrificed on the eighth day for relative quantitative detection of viral load by QPCR.Results:From 2004 to 2019,a total of 93,947 people in China were infected with dengue fever.Guangdong and Yunnan provinces are the main provinces through which dengue virus strains are imported from abroad,and there are certain differences in the sequence of the imported virus.In 2019,a dengue fever pandemic broke out in Xishuangbanna,Yunnan.DENV-1 infection and DENV-2 and DENV-3 infections coexisted in this epidemic.Among the 50 virus samples,34 cases of DENV-1 infection,15 cases of DENV-2 infection and 1 case of DENV-3 infection were detected by typing.The highest proportion of DENV-1 was selected for sequence analysis,and the full-length gene sequences of 8 strains of DENV-1 were obtained in the amplification and sequencing of the whole gene.Among them,7 DENY-1 epidemic strains were closely related to the dengue virus strains from Myanmar,Thailand,Laos and other countries in 2019,but were far related to the dengue virus strains from Jinghong City,Xishuangbanna Dai Autonomous Prefecture in 2015 and 2017.The wild strains DENV-1 and DENV-2 extracted from patients' serum showed three meaningful mutations on the amino acid site compared with the attenuated strains obtained after gradient cooling.After dengue virus was concentrated and purified,the TCID50 titer was measured as DENV-1 wild strain(1.64x107PFU/ml),DENV-1 attenuated strain(1.33x107PFU/ml),DENV-2 wild strain(1.92x107PFU/ml),DENV-2 attenuated strain(1.01x107PFU/ml).The concentrated and purified virus was diluted and subcutaneously injected into 2-week-old AG 129(IFN-???R-/-)mice at a dose of 1x104PFU to detect daily changes in body temperature and body weight.The results of Q-PCR relative quantitative detection of viral load in whole blood showed that there are differences in the change of virus copy number in the wild and attenuated strains of DENV-1 and DENV-2,and the time point of the highest virus copy number is different.The relative viral load in the organs of the mice on day 8 showed that the virus copy number was highest in the spleen,higher in the liver,and lower in the brain.The pathological changes of spleen were found not significant,but spleen enlargement appeared in some of the Dengue virus injected mice.The liver injury was more obvious in the pathological section,and the inflammatory cell infiltration and hepatocyte empties of the DENV-1 wild strain were stronger than those of the DENV-1 candidate attenuated strain.Contrary to expectations,DENV-2 candidate attenuated strain showed stronger effects on weight loss and liver damage in AG 129 young mice than the wild DENV-2 strain.Conclusion:The imported dengue virus strain was the main virus epidemic in the dengue fever outbreak in Xishuangbanna in 2019,and and it was introduced to the Southeast Asian countries on Indo-China Peninsula.The epidemic strains of dengue fever in Jinghong City in 2015 and 2017 were not the main epidemic strains of dengue fever in 2019.The attenuated candidate DENV-1 strain for low temperature adaptation showed weaker virulence to young mice than the wild strain,suggesting that the attenuated culture method had a certain effect on the virulence of Dengue virus.However,the attenuated candidate DENV-2 strain needs to be further evaluated and analyzed. |
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